Principles of Virology, Volume 1. Jane Flint

and experimental samples. Quantitative PCR is widely used in research and clinical applications for genotyping, gene expression analysis, copy number variation assays, and pathogen detection. While PCR is often used to detect viral genomes in clinical specimens or during experimental research, it is important to recognize that the nucleic acid detected does not necessarily correspond to infectious virus (Box 2.8).

BOX 2.8

      EXPERIMENTS

       Viral RNA is not infectious virus

      A study of sexual transmission of Zika virus among mice demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus particles.

      Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid, by PCR to detect viral RNA and by plaque assay to detect infectious virus particles. The results (see figure) show that Zika virus RNA persisted in semen for up to 60 days, far longer than did infectious virus, which could not be detected after about three weeks.

      There is a lower limit of detection of virus via the plaque assay of approximately 10 plaque forming units/ml. Whether this low concentration of infectious particles would be sufficient to transmit the virus is not known. However, it seems unlikely that these mice are able to transmit virus after a few weeks, despite the presence of Zika virus RNA in seminal fluid for at least 60 days after infection.

      Recently many papers have been published demonstrating that Zika virus and Ebolavirus can persist in a variety of human fluids for extended periods of time. These results have been interpreted with alarm by both by scientists and science writers. However, in most cases detection was by PCR, not by plaque assay, and therefore, we do not know if infectious virus particles were present. Viral RNA would not constitute a threat to transmission, while infectious virus would.

      Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood—it detects nucleic acids, not infectious virus.

      The lesson from this study is very clear: in novel experimental or epidemiological studies it is important to prove that any viral nucleic acid detected by PCR represents infectious virus. Failing to do so clouds the conclusions of the study.

      Detection of Zika virus RNA and infectious virus in seminal fluid. Male mice were infected with Zika virus. At different times after infection, viral RNA and infectious virus particles were measured in seminal fluid by PCR (blue line) and by plaque assay (red line).

       Duggal NK, Ritter JM, Pestorius SE, Zaki SR, Davis BS, Chang GJ, Bowen RA, Brault AC. 2017. Frequent Zika virus sexual transmission and prolonged viral RNA shedding in an immunodeficient mouse model. Cell Rep 18:1751–1760.

      High-throughput sequencing. The development of DNA sequencing methods in the 1970s revolutionized biology by allowing the decoding of viral genes and entire viral genomes. While powerful, these methods were laborious: in 1980 it took one year for a single person to determine the nucleotide sequence of the 7,440-nucleotide genome of poliovirus. Today the same result could be achieved in less than one hour.

      The difference is a consequence of the development of second- and third-generation sequencing methods, spurred by the desire to sequence larger and larger virus and cell genomes. These methods were originally called next-generation sequencing, because they followed the very first sequencing methods. The first of these new methods to be developed, 454 sequencing, was released in 2005 and could produce 200,000 reads of 110 base pairs. Other technologies that generated larger numbers of sequence reads soon followed (Solexa/ Illumina, SOLiD, and Ion Torrent) which generated larger numbers of reads, but the number of bases in each read was much shorter. These technologies relied on amplification of the target DNA and optical detection of incorporated fluorescent nucleotides. Third-generation sequencing methods can not only detect single molecules (e.g., amplification is not required) but also carry out sequencing in real time. PacBio instruments can achieve maximum read lengths of 20 kb, and those from Illumina can generate 1.8 terabytes of sequence per run. The latter reduces the cost of sequencing a human genome to below $1,000, a 10,000-fold reduction in price since 2004, when the first human genome was deciphered.

These technologies have not only made sequencing of DNA cheaper and faster but also helped create innovative experimental approaches to study genome organization, function, and evolution. Their use has led to the discovery of new viruses and has given birth to the field of metagenomics, the analysis of sequences directly from clinical or environmental samples. These sequencing technologies can be used to study the virome, the genomes of all viruses in a specific environment, such as sewage, the human body, or the intestinal tract. While these virus detection technologies are extremely powerful, the results obtained must be interpreted with caution. It is very easy to detect traces of a viral contaminant when searching for new agents of human disease (Box 2.9).

      It should be noted that metagenomics is not limited to DNA viruses. Nucleic acids extracted from clinical or environmental samples may be treated with DNase, and the remaining RNAs converted to DNA with reverse transcriptase for sequencing and identification.

BOX 2.9

      EXPERIMENTS

       Pathogen de-discovery

      High-throughput sequencing of nucleic acids has accelerated the pace of virus discovery, but at a cost: contaminants are much easier to detect.

      During a search for the causative agent of seronegative hepatitis (disease not caused by hepatitis A, B, C, D, or E virus) in Chinese patients, a new virus with a single-stranded DNA genome was discovered in sera by high-throughput sequencing. Seventy percent of 90 patient serum samples were positive for viral DNA by PCR, and sera from 45 healthy controls were negative. Furthermore, 84% of patients were positive for antibodies against the virus. Among healthy controls, 78% were antibody positive. The authors concluded that this virus was highly prevalent in some patients with seronegative hepatitis. A second independent laboratory identified the same virus in sera from patients in the United States with non-A-to-E hepatitis, while a third group identified the virus in diarrheal stool samples from Nigeria.

      The first clue that something was amiss was the observation that the new virus identified in all three laboratories shared 99% nucleotide and amino acid identity: this similarity would not be expected in virus samples from such geographically, temporally, and clinically diverse samples. Another problem was that in the U.S. non-A-to-E hepatitis study, all pools of patient sera were positive for viral sequences. These observations suggested the possibility of viral contamination.

      When nucleic acids were repurified from the U.S. non-A-to-E hepatitis samples using a different method, none were positive for the new virus. The presence of the virus was traced to the use of column-based purification kits manufactured by Qiagen, Inc. (pictured). Nearly the entire viral genome could be detected by deep sequencing of sterile water that was passed through these columns. The nucleic acid purification columns contaminated with the new virus were used to purify nucleic acid from patient samples. These columns, produced by a number of manufacturers, are typically an inch in length and contain a silica gel membrane that binds nucleic acids. The clinical samples are added to the column,